Secretagogin Immunoreactivity Reveals Lugaro Cells in the Pigeon Cerebellum.

Lugaro cells are inhibitory interneurons found in the upper granular layer of the cerebellar cortex, just below or within the Purkinje cell layer. They are characterized by (1) a fusiform soma oriented in the parasagittal plane, (2) two pairs of dendrites emanating from opposite ends of the soma, (3) innervation from Purkinje cell collaterals, and (4) an axon that projects into the molecular layer akin to granular cell parallel fibers. Lugaro cells have been described in mammals, but not in other vertebrate classes, save one report in teleost fish. Here, we propose the existence of Lugaro cells in the avian cerebellum based on the morphological characteristics and connectivity described above. Immunohistochemical staining against the calcium binding protein secretagogin (SCGN) revealed Lugaro-like cells in the pigeon cerebellum. Some SCGN-labeled cells exhibit fusiform somata and dendrites parallel to the Purkinje cell layer in the parasagittal plane, as well as long axons that project into the molecular layer and travel alongside parallel fibers in the coronal plane. While mammalian Lugaro cells are known to express calretinin, the SCGN-labeled cells in the pigeon do not. SCGN-labeled cells also express glutamic acid decarboxylase, confirming their inhibitory function. Calbindin labeling revealed Purkinje cell terminals surrounding the SCGN-expressing cells. Our results suggest that Lugaro cells are more widespread among vertebrates than previously thought and may be a characteristic of the cerebellum of all vertebrates.

Is Cerebellar Architecture Shaped by Sensory Ecology in the New Zealand Kiwi (Apteryx mantelli).

Among some mammals and birds, the cerebellar architecture appears to be adapted to the animal's ecological niche, particularly their sensory ecology and behavior. This relationship is, however, not well understood. To explore this, we examined the expression of zebrin II (ZII) in the cerebellum of the kiwi (Apteryx mantelli), a fully nocturnal bird with auditory, tactile, and olfactory specializations and a reduced visual system. We predicted that the cerebellar architecture, particularly those regions receiving visual inputs and those that receive trigeminal afferents from their beak, would be modified in accordance with their unique way of life. The general stripe-and-transverse region architecture characteristic of birds is present in kiwi, with some differences. Folium IXcd was characterized by large ZII-positive stripes and all Purkinje cells in the flocculus were ZII positive, features that resemble those of small mammals and suggest a visual ecology unlike that of other birds. The central region in kiwi appeared reduced or modified, with folium IV containing ZII+/- stripes, unlike that of most birds, but similar to that of Chilean tinamous. It is possible that a reduced visual system has contributed to a small central region, although increased trigeminal input and flightlessness have undoubtedly played a role in shaping its architecture. Overall, like in mammals, the cerebellar architecture in kiwi and other birds may be substantially modified to serve a particular ecological niche, although we still require a larger comparative data set to fully understand this relationship.

Zebrin II Expression in the Cerebellum of a Paleognathous Bird, the Chilean Tinamou (Nothoprocta perdicaria).

Zebrin II (ZII) is a glycolytic enzyme expressed in cerebellar Purkinje cells. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with a high ZII expression (ZII+) alternating with stripes of Purkinje cells with little or no expression (ZII-). To date, ZII expression studies are limited to neognathous birds: pigeons (Columbiformes), chickens (Galliformes), and hummingbirds (Trochilidae). These previous studies divided the avian cerebellum into 5 transverse regions based on the pattern of ZII expression. In the lingular region (lobule I) all Purkinje cells are ZII+. In the anterior region (lobules II-V) there are 4 pairs of ZII+/- stripes. In the central region (lobules VI-VIII) all Purkinje cells are ZII+. In the posterior region (lobules VIII-IX) there are 5-7 pairs of ZII+/- stripes. Finally, in the nodular region (lobule X) all Purkinje cells are ZII+. As the pattern of ZII stripes is quite similar in these disparate species, it appears that it is highly conserved. However, it has yet to be studied in paleognathous birds, which split from the neognaths over 100 million years ago. To better understand the evolution of cerebellar compartmentation in birds, we examined ZII immunoreactivity in a paleognath, the Chilean tinamou (Nothoprocta perdicaria). In the tinamou, Purkinje cells expressed ZII heterogeneously such that there were sagittal ZII+ and ZII- stripes of Purkinje cells, and this pattern of expression was largely similar to that observed in neognathous birds. For example, all Purkinje cells in the lingular (lobule I) and nodular (lobule X) regions were ZII+, and there were 4 pairs of ZII+/- stripes in the anterior region (lobules II-V). In contrast to neognaths, however, ZII was expressed in lobules VI-VII as a series of sagittal stripes in the tinamou. Also unlike in neognaths, stripes were absent in lobule IXab, and all Purkinje cells expressed ZII in the tinamou. The differences in ZII expression between the tinamou and neognaths could reflect behavior, but the general similarity of the expression patterns across all bird species suggests that ZII stripes evolved early in the avian phylogenetic tree.

Topographic Organization of Inferior Olive Projections to the Zebrin II Stripes in the Pigeon Cerebellar Uvula.

This study was aimed at mapping the organization of the projections from the inferior olive (IO) to the ventral uvula in pigeons. The uvula is part of the vestibulocerebellum (VbC), which is involved in the processing of optic flow resulting from self-motion. As in other areas of the cerebellum, the uvula is organized into sagittal zones, which is apparent with respect to afferent inputs, the projection patterns of Purkinje cell (PC) efferents, the response properties of PCs and the expression of molecular markers such as zebrin II (ZII). ZII is heterogeneously expressed such that there are sagittal stripes of PCs with high ZII expression (ZII+), alternating with sagittal stripes of PCs with little to no ZII expression (ZII-). We have previously demonstrated that a ZII+/- stripe pair in the uvula constitutes a functional unit, insofar as the complex spike activity (CSA) of all PCs within a ZII+/- stripe pair respond to the same type of optic flow stimuli. In the present study we sought to map the climbing fiber (CF) inputs from the IO to the ZII+ and ZII- stripes in the uvula. We injected fluorescent Cholera Toxin B (CTB) of different colors (red and green) into ZII+ and ZII- bands of functional stripe pair. Injections in the ZII+ and ZII- bands resulted in retrograde labeling of spatially separate, but adjacent regions in the IO. Thus, although a ZII+/- stripe pair represents a functional unit in the pigeon uvula, CF inputs to the ZII+ and ZII- stripes of a unit arise from separate regions of the IO.

Inferior olivary projection to the zebrin II stripes in lobule IXcd of the pigeon flocculus: A retrograde tracing study.

Zebrin II (ZII; a.k.a. aldolase C) is expressed heterogeneously in Purkinje cells (PCs) such that there are sagittal stripes of high expression (ZII+) interdigitated with stripes of little or no expression (ZII-). The pigeon flocculus receives visual-optokinetic information and is important for generating compensatory eye movements. It consists of 4 sagittal zones based on PC complex spike activity (CSA) in response to rotational optokinetic stimuli. There are two zones where CSA responds best to rotation about the vertical axis (VA), interdigitated with two zones where CSA responds best to rotation about an horizontal axis (HA). These optokinetic zones relate to the ZII stripes in folium IXcd of the flocculus, such that an optokinetic zone spans a ZII+/- pair: the HA zones span the P5+/- and P7+/- ZII stripe pairs, whereas the VA zones correspond to ZII stripe pairs P4+/- and P6+/-. In the present study, we used fluorescent retrograde tracing to determine the olivary inputs to the ZII+ and ZII- stripes within the functional pairs. We found that separate but adjacent areas of the medial column of the inferior olive (mcIO) project to the ZII+ and ZII- stripes within each of the functional pairs. Thus, although a ZII+/- stripe pair represents a functional unit in the pigeon flocculus insofar as the CSA of all PCs in the stripe pair encodes similar sensory information, the olivary inputs to the ZII+ and ZII- stripes arise from different, although adjacent, regions of the mcIO.